Biology subjects

Romero-Perez, L.

Publications and source records attributed to Romero-Perez, L..

12 recordsLinked to original sources

bioRxiv2025

Targeting CXADR-mediated AKT signaling suppresses tumorigenesis and enhances chemotherapy efficacy in Ewing sarcoma

Distant metastasis is the leading cause of mortality in Ewing sarcoma (EwS) - a malignant bone or soft-tissue cancer mainly affecting children, adolescents, and young adults. Despite continuous efforts in understanding its pathogenesis, the molecular mechanisms driving EwS metastasis remain poorly understood, thus limiting the potential for therapeutic progress. Here, we identify the tight junction component Coxsackievirus and Adenovirus receptor (CXADR) as a critical regulator of cancer progression and metastasis in EwS. Differential gene expression analysis of patient tumors from two independent cohorts revealed that elevated CXADR levels are associated with metastatic disease and poor overall survival. In functional experiments, conditional CXADR knockdown reduced the growth of EwS cell line models in vitro, and suppressed local tumorigenesis. Notably, CXADR knockdown completely abrogated metastasis formation in vivo. Integration of transcriptome profiling and mechanistic studies uncovered that CXADR promotes the activation of AKT signaling, likely through complex formation with PTEN. Consequently, pharmacological targeting of AKT using the FDA-approved pan-AKT inhibitor Capivasertib showed CXADR-dependent cytotoxicity, with enhanced efficacy if combined with the EwS standard-of-care chemotherapeutic agent Vincristine. Collectively, our findings establish CXADR as a prognostic and predictive biomarker in EwS, highlighting AKT inhibition combined with chemotherapy as a promising strategy for patients with high CXADR expression. Together, these findings support a precision medicine approach combining molecular stratification and targeted therapies to improve patient outcomes in metastatic EwS.

cancer biology↗

bioRxiv2024

Super-enhancer-driven CACNA2D2 is an EWSR1::WT1 signature gene encoding a diagnostic marker for desmoplastic small round cell tumor (DSRCT)

Desmoplastic small round cell tumor (DSRCT) is a highly aggressive cancer predominantly occurring in male adolescents and young adults. The lack of a comprehensive understanding on the biology of the disease is paralleled by its dismal survival rates (5-20%). To overcome this challenge, we first identified and prioritized urgently needed resources for clinicians and researchers. Thus, we established genome-wide single-cell RNA-sequencing and bulk proteomic data of in vitro and in vivo-generated knockdown models of the pathognomonic DSRCT fusion oncoprotein (EWSR1::WT1) and combined them with an original systems-biology-based pipeline including patient data and the largest histology collection of DSRCTs and morphological mimics available to date. These novel tools were enriched with curated public datasets including patient- and cell line-derived ChIP-seq, bulk and single-cell RNA-seq studies resulting in a multi-model and multi-omic toolbox for discovery analyses. As a proof of concept, our approach revealed the alpha-2/delta subunit of the voltage-dependent calcium channel complex, CACNA2D2, as a highly overexpressed, super-enhancer driven, direct target of EWSR1::WT1. Single-cell and bulk-level analyses of patient samples and xenografted cell lines highlighted CACNA2D2 as a critical component of our newly established EWSR1::WT1 oncogenic signature, that can be employed to robustly identify DSRCT in reference sets. Finally, we show that CACNA2D2 is a highly sensitive and specific single biomarker for fast, simple, and cost-efficient diagnosis of DSRCT. Collectively, we establish a large-scale multi-omics dataset for this devastating disease and provide a blueprint of how such toolbox can be used to identify new and clinically relevant diagnostic markers, which may significantly reduce misdiagnoses, and thus improve patient care.

cancer biology↗

bioRxiv2022

Chromosome 8 gain drives poor patient outcome via expression of 4E-BP1 in Ewing sarcoma

Chromosome 8 (chr8) gains are common in cancer. However, their potential contribution to tumor heterogeneity is largely unexplored. Ewing sarcoma (EwS) is characterized by pathognomonic FET::ETS fusions but a general paucity of other recurrent somatic mutations that could explain the observed clinical diversity. In EwS, chr8 gains are the second most common genetic alteration rendering EwS an ideal model to investigate the relevance of chr8 gains in an otherwise silent genomic context. Here, we report that chr8 gain-driven gene expression patterns correlate with poor overall survival of EwS patients. This effect is predominantly mediated by increased expression of the translation initiation factor binding protein 4E-BP1 encoded by EIF4EBP1 on chr8. High EIF4EBP1 expression showed the strongest association with poor patient survival among all chr8-encoded genes and correlated with chr8 gains in EwS tumors. Similar findings were made in numerous entities of The Cancer Genome Atlas (TCGA). Integrated multi-omics profiling uncovered that 4E-BP1 orchestrates a pro-proliferative proteomic network. Consistently, silencing of 4E-BP1 in the EwS model reduced cell proliferation, clonogenicity, spheroidal growth in vitro, and tumorigenesis in vivo. Drug screens and functional assays revealed that high 4E-BP1 expression sensitizes for pharmacological CDK4/6 inhibition in preclinical models. Collectively, we establish chr8 gains and high 4E-BP1 expression as prognostic biomarkers in EwS and demonstrate that their association with patient outcome is primarily mediated by 4E-BP1 orchestrating a pro-proliferative proteomic network sensitizing EwS for CDK4/6 inhibitors. Our data suggest that testing for chr8 gains may improve risk-stratification and therapeutic management in EwS and other cancers.

cancer biology↗

bioRxiv2021

Translational evidence for RRM2 as a prognostic biomarker and therapeutic target in Ewing sarcoma

PurposeEwing sarcoma (EwS) is a highly aggressive bone- or soft tissue-associated malignancy mostly affecting children, adolescents, and young adults. Although multimodal therapies have strongly improved patients overall survival over the past decades, the development of prognostic biomarkers for risk-based patient stratification and more effective therapies with less adverse effects is stagnating. Thus, new personalized medicine approaches are urgently required. Experimental designGene expression data of EwS and normal tissues were crossed with survival data to identify highly overexpressed, prognostically relevant, and actionable potential targets. RNA-interference and dose-response assays as well as tissue-microarray analyses were carried out to explore the functional role and druggability of a prominent candidate gene in vitro and in vivo, and to validate its suitability as a prognostic biomarker. ResultsEmploying a multilayered screening approach, we discover ribonucleotide reductase regulatory subunit M2 (RRM2) as a promising therapeutic target and prognostic biomarker in EwS. Through analysis of two independent EwS patient cohorts, we show that RRM2 mRNA and protein overexpression is associated with an aggressive clinical phenotype and poor patients overall survival. In agreement, RRM2 silencing as well as pharmacological inhibition by the specific inhibitor triapine (3-AP) significantly reduces EwS growth in vitro and in vivo. Furthermore, we present evidence that pharmacological RRM2 inhibition by triapine can overcome chemoresistance against doxorubicin or gemcitabine, and synergize with cell cycle checkpoint inhibitors (CHEK1 or WEE1). ConclusionsBased on the aggressive phenotype mediated by and the druggability of RRM2 our results provide a translational rationale for exploiting RRM2 as a novel therapeutic target in EwS and prompt further clinical investigations.

cancer biology↗

bioRxiv2021

Systems biology analysis identifies TCF7L1 as a key regulator of metastasis in Ewing sarcoma

Identification of cancer stemness genes is crucial to understanding the underlying biology of therapy resistance, relapse, and metastasis. Ewing sarcoma (EwS) is the second most common bone tumor in children and adolescents. It is a highly aggressive cancer associated with a dismal survival rate (<30%) for patients with metastatic disease at diagnosis ([~]25% of cases). Hence, deciphering the underlying mechanisms of metastasis is imperative. EwS tumors are characterized by a remarkably silent genome with a single driver mutation generating an oncogenic fusion transcription factor (EWSR1-ETS). Thus, EwS constitutes an ideal model to study how perturbation of a transcriptional network by a dominant oncogene can mediate metastasis, even though canonical metastasis-associated genes are not mutated. Here, through the implementation of an integrative systems biology approach, we identified transcription factor 7 like 1 (TCF7L1, alias TCF3) as a prognostically-relevant and EWSR1-ETS suppressed determinant of metastasis in EwS. We demonstrated that conditional TCF7L1 re-expression significantly reduces EwS single-cell migration, invasion and anchorage-independent growth in 3D assays in vitro, and tumorigenesis in vivo mediated by its DNA binding domain. In primary EwS tumors as well as in functional orthotopic in vivo models, low TCF7L1 expression was associated with pro-metastatic gene signatures and a much higher migratory and metastatic capacity of EwS cells, which correlated with poor outcome of EwS patients. Collectively, our findings establish TCF7L1 as a major regulator of metastasis in EwS, which may be utilized as a prognostic biomarker and open inroads to future therapeutic intervention.

cancer biology↗

bioRxiv2019

Hippo pathway effectors YAP1/TAZ induce a EWS-FLI1-opposing gene signature and associate with disease progression in Ewing Sarcoma

YAP1 and TAZ (WWTR1) oncoproteins are the final transducers of Hippo tumor suppressor pathway. Deregulation of the pathway leads to YAP1/TAZ activation fostering tumorigenesis in multiple malignant tumor types, including sarcoma. However, oncogenic mutations within the core components of the Hippo pathway are uncommon. Ewing Sarcoma (EwS), a pediatric cancer with low mutation rate, is characterized by a canonical fusion involving EWSR1 gene, and FLI1 as the most common partner. The fusion protein is a potent driver of oncogenesis but secondary alterations are scarce, and little is known about other biological factors that determine the risk of relapse or progression. We have observed YAP1/TAZ expression and transcriptional activity in EwS cell lines. Analyses of 55 primary human EwS samples revealed that high YAP1/TAZ expression was associated with progression of the disease and predicted poorer outcome.\n\nWe did not observe recurrent SNV or copy number gains/losses in Hippo pathway-related loci. However, differential CpG methylation of RASSF1 locus -a regulator of Hippo pathway- was observed in EwS cell lines compared with mesenchymal stem cells, the putative cell of origin of EwS. Hypermethylation of RASSF1 correlated with the transcriptional silencing of the tumor suppressor isoform RASFF1A, and transcriptional activation of the protumorigenic isoform RASSF1C promoting YAP1/TAZ activation. Knockdown of YAP1/TAZ decreased proliferation and invasion abilities of EwS cells, and revealed that YAP1/TAZ transcription activity is inversely correlated with the EWS-FLI1 transcriptional signature. This transcriptional antagonism could be partly explained by EWS-FLI1-mediated transcriptional repression of TAZ. Thus, YAP1/TAZ may override the transcriptional program induced by the fusion protein, contributing to the phenotypic plasticity determined by dynamic fluctuation of the fusion protein, a recently proposed model for disease dissemination in EwS.

cancer biology↗

bioRxiv2019

Oncogenic hijacking of a developmental transcription factor evokes therapeutic vulnerability for ROS-induction in Ewing sarcoma

Ewing sarcoma (EwS) is an aggressive childhood cancer likely originating from mesenchymal stem cells or osteo-chondrogenic progenitors. It is characterized by fusion oncoproteins involving EWSR1 and variable members of the ETS-family of transcription factors (in 85% FLI1). EWSR1-FLI1 can induce target genes by using GGAA-microsatellites (mSats) as enhancers.\n\nHere, we show that EWSR1-FLI1 hijacks the developmental transcription factor SOX6 - a physiological driver of proliferation of osteo-chondrogenic progenitors - by binding to an intronic GGAA-mSat, which promotes EwS growth in vitro and in vivo. Through integration of transcriptome-profiling, published drug-screening data, and functional in vitro and in vivo experiments, we discovered that SOX6 interferes with the antioxidant system resulting in constitutively elevated reactive oxygen species (ROS) levels that create a therapeutic vulnerability toward the ROS-inducing drug Elesclomol.\n\nCollectively, our results exemplify how aberrant activation of a developmental transcription factor by a dominant oncogene can promote malignancy, but provide opportunities for targeted therapy.

cancer biology↗

bioRxiv2019

Expression patterns of PD-L1 and PD-1 provide rationales for immune checkpoint inhibition in soft tissue sarcomas

Soft tissue sarcomas (STS) are highly malignant cancers with mesenchymal origin. In many instances, clinical outcome is poor due to high rates of local recurrence and metastasis.\n\nThe programmed death receptor ligand 1 (PD-L1) is expressed in several cancers. PD-L1 interacts with its receptor, PD-1, on the surface of tumor infiltrating lymphocytes (TILs), thereby attenuating anti-cancer immune response. Immune checkpoint inhibitors targeting this interaction are promising new anti-cancer drugs. However, present studies on the PD-L1 and PD-1 expression status in STS are limited either by small sample size, analysis of single STS subtypes, or lack of combinatorial assessment of PD-L1, PD-1 and TILs.\n\nTo overcome these limitations, we evaluated the expression patterns of intratumoral PD-L1, the amount of TILs and their PD-1 expression status, as well as associations with clinicopathological parameters in a large and comprehensive cohort of 274 samples comprising more than six STS subtypes.\n\nWe found that nearly all STS subtypes showed partial PD-L1 expression, albeit with a broad range of PD-L1 positivity across subtypes (50% angiosarcomas, 23% UPS, 13% leiomyosarcomas, 12% dedifferentiated liposarcomas, 3% synovial sarcomas, 0 MPNST, and 18% mixed sarcomas). Co-expression and correlation analyses uncovered that expression of PD-L1 was associated with more PD-1 positive TILs (P < 0.001), higher tumor grading (P = 0.022) and worse patients 5-year overall survival (P = 0.016).\n\nIn sum, the substantial portion of STS showing PD-L1 expression, the simultaneous presence of PD-1 positive TILs, and the association of PD-L1 with unfavorable clinical outcome provide a rationale for immune checkpoint inhibition in patients with PD-L1-positive STS.

cancer biology↗

bioRxiv2019

Biomarker-based outcome prediction in prostate adenocarcinoma depends on the TMPRSS2-ERG status

BackgroundProstate adenocarcinoma (PCa) with/without the TMPRSS2-ERG (T2E)-fusion represent distinct molecular subtypes. ObjectiveTo investigate gene-signatures associated with metastasis in T2E-positive and -negative PCa, and to identify and validate subtype-specific prognostic biomarkers. Design, setting and participantsGene expression and clinicopathological data of two discovery PCa cohorts (total n=783) were separately analyzed regarding the T2E-status. Selected subtype-specific biomarkers were validated in two additional cohorts (total n=405). Outcome measurements and statistical analysisFrom both discovery cohorts, we generated two gene lists ranked by their differential intratumoral expression in patients with/without metastases stratified by T2E-status, which were subjected to gene set enrichment and leading-edge analyses. The resulting top 20 gene-signatures of both gene lists associated with metastasis were analyzed for overlaps between T2E-positive and -negative cases. Genes shared by several functional gene-signatures were tested for their association with event-free survival using the Kaplan-Meier method in a validation cohort. Immunohistochemistry was performed in another validation cohort. Results and limitationsMetastatic T2E-positive and -negative PCa are characterized by different gene-signatures. Five genes (ASPN, BGN, COL1A1, RRM2 and TYMS) were identified whose high expression was significantly associated with worse outcome exclusively in T2E-negative PCa. This was validated in an independent cohort for all genes and additionally for RRM2 by immunohistochemistry in a separate validation cohort. No prognostic biomarkers were identified exclusively for T2E-positive tumors. ConclusionsOur study demonstrates that the prognostic value of biomarkers critically depends on the molecular subtype, i.e. the T2E-status, which should be considered when screening for and applying novel prognostic biomarkers for outcome prediction in PCa. Patient summaryOutcome prediction for PCa is complex. The results of this study highlight that the validity of prognostic biomarkers depends on the molecular subtype, specifically the presence/absence of T2E. The reported new subtype-specific biomarkers exemplify that biomarker-based outcome prediction in PCa should consider the T2E-status.

cancer biology↗

bioRxiv2018

Cooperation of dominant oncogenes with regulatory germline variants shapes clinical outcomes in childhood cancer

INTRODUCTORY PARAGRAPHDeciphering principles of inter-individual tumor heterogeneity is essential for refinement of personalized anti-cancer therapy. Unlike cancers of adulthood, pediatric malignancies including Ewing sarcoma (EwS) feature a striking paucity of somatic alterations except for pathognomonic driver-mutations that cannot explain overt variations in clinical outcome.\n\nHere we demonstrate in the EwS model how cooperation of a dominant oncogene and regulatory variants determine tumor growth, patient survival and drug response.\n\nWe show that binding of the oncogenic EWSR1-FLI1 fusion transcription factor to a polymorphic enhancer-like DNA element controls expression of the transcription factor MYBL2, whose high expression promotes poor patient outcome via activation of pro-proliferative signatures. Analysis of paired germline and tumor whole-genome sequencing data revealed that regulatory variability at this locus is inherited via the germline. CRISPR-mediated interference with this regulatory element almost abolished MYBL2 transcription, and MYBL2 knockdown decreased cell proliferation, cell survival and tumorigenicity of EwS cells. Combined RNA- and ChIP-seq analyses as well as functional experiments and clinical data identified CCNF, BIRC5 and AURKB as direct MYBL2 targets and critical mediators of its phenotype. In drug-response experiments, high MYBL2 levels sensitized EwS cells for inhibition of its activating cyclin dependent kinase CDK2 in vitro and in vivo, suggesting MYBL2 as a predictive biomarker for targeted anti-CDK2-therapy.\n\nCollectively, our findings establish cooperation of somatic mutations and regulatory germline variants as a major determinant of tumor progression and indicate the importance of integrating the regulatory genome in the process of developing new diagnostic and/or therapeutic strategies to fully harness the potential of precision medicine.

cancer biology↗

bioRxiv2018

SOX2 expression identifies Ewing sarcoma patients with high risk for tumor relapse and poor survival

Purpose: Up to 30-40% of Ewing sarcoma (EwS) patients with non-metastatic disease develop local or metastatic relapse within a time range of 2-10 years. This is in part caused by the absence of prognostic biomarkers that can identify high-risk patients to assign them to risk-adapted monitoring and treatment regimens. Since cancer stemness has been associated with tumor relapse and poor outcome, we investigated in the current study the prognostic potential SOX2 (sex determining region Y box 2) - a major transcription factor involved in development and stemness - previously described to contribute to the undifferentiated phenotype of EwS. Methods: Two independent patient cohorts, one consisting of 189 retrospectively collected EwS tumors with corresponding mRNA expression data (test cohort) and the other of 141 prospectively collected formalin-fixed and paraffin embedded resected tumors (validation cohort), were employed to analyze SOX2 expression levels by DNA microarrays or immunohistochemistry, respectively, and to compare them with clinical parameters and patient outcome. Two methods were employed to test the validity of the results at both mRNA and protein levels. Results: Both cohorts showed that only a subset of EwS patients (16-20%) express high SOX2 mRNA or protein levels, which significantly correlated with poor overall survival. Multivariate analyses of our validation cohort revealed that high SOX2 expression represents a major risk factor for survival (HR=3.19; P<0.01) that is independent from metastasis and other known clinical risk-factors at time of diagnosis. Univariate analysis demonstrated that SOX2-high expression correlated with tumor relapse (P=0.001). Median first relapse was at 14.7 months (range: 3.5-180.7). Conclusions: High SOX2 expression constitutes an independent prognostic biomarker for EwS patients with poor outcome, which may help to identify patients with localized disease who are at high risk for tumor relapse within the first two years after diagnosis.

pathology↗

bioRxiv2018

Targeting the CALCB/RAMP1-axis inhibits growth of Ewing sarcoma

Ewing sarcoma (EwS) is an aggressive cancer caused by chromosomal translocations generating fusions of the EWSR1 gene with ETS transcription factors (in 85% FLI1). EWSR1-FLI1 induces gene expression via binding to enhancer-like GGAA-microsatellites, whose activity increases with the number of consecutive GGAA-repeats.\n\nHerein, we investigate the role of the secretory neuropeptide CALCB (calcitonin related polypeptide {beta}) in EwS, which signals via the CGRP-(calcitonin gene-related peptide) receptor complex, containing RAMP1 (receptor activity modifying protein 1) as crucial part for receptor specificity. Analysis of 2,678 gene expression microarrays comprising 50 tumor entities and 71 normal tissue types revealed that CALCB is specifically and highly overexpressed in EwS. Time-course knockdown experiments showed that CALCB expression is tightly linked to that of EWSR1-FLI1. Consistently, gene set enrichment analyses of genes whose expression in primary EwS is correlated to that of CALCB indicated that it is co-expressed with other EWSR1-FLI1 target genes and associated with signatures involved in stemness and proliferation. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) data for EWSR1-FLI1 and histone marks from EwS cells demonstrated that EWSR1-FLI1 binds to a GGAA-microsatellite close to CALCB, which exhibits characteristics of an active enhancer. Reporter assays confirmed the strong EWSR1-FLI1- and length-dependent enhancer activity of this GGAA-microsatellite. Mass-spectrometry analyses of supernatants of EwS cell cultures demonstrated that CALCB is secreted by EwS cells. While short-term RNA interference-mediated CALCB knockdown had no effect on proliferation and clonogenic growth of EwS cells in vitro, its long-term knockdown decreased EwS growth in vitro and in vivo. Similarly, knockdown of RAMP1 reduced clonogenic/spheroidal growth and tumorigenicity, and small-molecule inhibitors directed against the CGRP-receptor comprising RAMP1 reduced growth of EwS.\n\nCollectively, our findings suggest that CALCB is a direct EWSR1-FLI1 target and that targeting the CALCB/RAMP1-axis may offer a new therapeutic strategy for inhibition of EwS growth.

cancer biology↗